Estradiol Assay

Concentrations of estradiol in plasma and FSH in serum were measured at baseline and end point of benzoate or placebo treatment. Estradiol protein concentrations were measured using commercially available enzyme-linked immunosorbent assay kits according to the manufacturer’s recommended protocol (DRG International). Briefly, 100 μL of plasma samples or the standard was added to each well of a 96-well plate, and 200 μL of enzyme-conjugate was then incubated for 4 hours at room temperature. The liquid was then removed. Each well was washed 3 times with wash buffer. Thereafter, 200 μL of substrate solution was added to each well and then incubated for 30 minutes at room temperature with protection from the light. Finally, 100 μL of stop solution was added to each well, and thorough mixing was ensured. The absorbance at 450 nm was assessed with the Benchmark Plus Microplate Reader (Bio-Rad), and the concentrations of estradiol in the samples were determined using a standard curve.